Nitric oxide (NO) is continuously synthesized by the endothelium, and contributes importantly to vasodilator tone of the coronary and systemic circulations by activating guanylyl cyclase in vascular smooth muscle, causing relaxation. Although regional synthesis of NO by the endothelium contributes to local vasodilator tone, Stamler and co-workers have proposed that regional vascular tone may also be regulated by NO transported from the lungs to distant vascular sites by hemoglobin and other proteins as a consequence of enhanced binding of NO to reactive thiol groups. In order to determine the contribution of blood-transported NO to regional vascular tone in humans, we measured forearm blood flow and calculated resistance (mean blood pressure/flow) in 16 healthy subjects (8 men, 8 women, average age 33 years) at rest and during regional hypoxia associated with forearm exercise stress, with measurements made before and after blockade of forearm endothelial NO synthesis. To this end, forearm blood flow was measured by strain gauge venous occlusion plethysmography, before and after inhibition of NO synthesis in the forearm with infusion of NG-monomethyl-L-arginine (L-NMMA; 8 mol/min), which competes with L-arginine as the substrate for NO synthase but cannot be oxidized to form NO. Infusion of L-NMMA at this dosage into the brachial artery avoids systemic effects of this NO synthase inhibitor, which might in turn have reflexive secondary effects on regional vascular tone. While breathing room air, intra-arterial L-NMMA for 5 minutes reduced forearm blood flow from 2.6+/-0.2 (mean+/-SEM) to 1.8+/-0.1 ml/min/100 g tissue (P=0.001). This 25% average reduction in forearm blood flow was associated with a 50% increase in forearm vascular resistance (34.1+/-3.1 to 48.3+/-3.9 resistance units, P=0.001) and with widening of the brachial artery minus brachial vein (A-V) pH difference (from 0.029+/-0.008 to 0.049+/-0.010 pH units, P=0.03) and widening of the A-V pO2 difference (from 59+/-4 to 64+/-4 mm Hg, P=0.04). After 5 minutes of repetitive hand-grip exercise during continued L-NMMA infusion, forearm blood flow increased to 19.9+/-2.2 ml/min/100 g tissue (P<0.001 vs. baseline). This 742% average increase in forearm blood flow was associated with an 84% reduction in forearm vascular resistance (to 5.0+/-0.6 resistance units, P<0.001 vs. baseline) and further widening of the A-V pH difference (to 0.124+/-0.012 pH units, P<0.001 vs. baseline) and the A-V pO2 difference (to 79+/-2 mm Hg, P<0.001 vs. baseline). Following termination of exercise, L-NMMA infusion was discontinued and inhalation of NO at 80 ppm was initiated, with on-line monitoring to assure continuous oxygen delivery at 21%. One hour later and with continuation of NO inhalation, the sequence of measurements described above on room air was repeated. During NO inhalation, intra-arterial infusion of L-NMMA at the same dosage no longer caused significant reduction in forearm blood flow (from 2.2+/-0.2 to 2.0+/-0.2 ml/min/100 g tissue, P=0.08). This 7% average reduction in forearm blood flow during L-NMMA infusion was significantly less than the 25% reduction in forearm blood flow achieved on room air (P=0.04). Similarly, the 11% average increase in forearm vascular resistance was significantly less than the 50% increase in resistance achieved on room air (P=0.018).During repetitive hand-grip exercise with continuation of intra-arterial L-NMMA infusion and NO inhalation, forearm blood flow increased to 21.0+/-2.0 ml/min/100 tissue. This 914% average increase in forearm blood flow tended to be greater than the 742|% increase in flow on room air (P=0.09); the 88% decrease in forearm vascular resistance was significantly greater than the 84% decrease in resistance on room air (P=0.01). Additionally, the A-V pH difference during exercise during L-NMMA infusion with NO inhalation was significantly less than room air exercise values (0.078+/-0.015 vs. 0.124+/-0.012 pH units, P<0.001), with nonsignificant reduction in the A-V pO2 difference (76+/-2 vs.79+/-2 mm Hg, P=0.08). In the contralateral arm, which did not receive intra-arterial L-NMMA infusion and thus served as the control arm, NO inhalation did not alter basal (2.6+/-0.3 vs. 2.7+/-0.4 ml/min/100 g tissue, P=0.875) or exercise (21.8+/-2.0 vs. 20.7+/-1.8 ml/min/100 g tissue, P=0.953) forearm blood flow compared with room air values, nor was there any effect of NO inhalation on A-V pH or pO2 differences compared with room air measurements. In order to determine the mechanism of bioactive NO delivery in blood during NO breathing, we measured all known NO species in arterial and venous blood of the forearm. S-nitrosohemoglobin and plasma S-nitrosothiols did not change with NO breathing. Arterial nitrite levels increased by 11% and arterial nitrosyl(heme)hemoglobin increased 4-fold to the micromolar range, and both measures were consistently higher in the arterial than in venous blood. We conclude that 1) regional inhibition of NO synthesis causes significant reduction in regional blood flow, indicating the importance of regional synthesis of NO in regulating local vascular tone, 2) NO inhalation a) significantly attenuates the reduction in forearm blood flow seen with regional blockade of NO synthesis, but with lesser effect on tissue oxygenation, b) significantly enhances the forearm blood flow response to exercise stress with less tissue acidosis, but c) has no effect on forearm blood flow at rest or during exercise in the presence of normal regional NO synthesis, and 3) nitrosyl(heme)hemoglobin and nitrite are potential transporters of bioactive NO to vascular tissue. These findings may be relevant to understanding the physiological contribution and therapeutic potential of hemoglobin-transported NO in the regulation of vasodilator tone in diseases and conditions associated with regional endothelial dysfunction and reduced endothelial NO bioactivity.